PL35 family chondroitin sulfate lyase CHa3 as well as coding gene and application thereof

Abstract

The invention relates to a PL35 family chondroitin sulfate lyase CHa3 as well as a coding gene and application thereof, and belongs to the technical field of enzyme engineering. The amino acid sequence of the chondroitin sulfate lyase CHa3 is shown as SEQ ID NO.2, and the chondroitin sulfate lyase CHa3 is novel chondroitin sulfate lyase, has high enzymatic activity on chondroitin sulfate, and degrades chondroitin sulfate into an incision enzyme mode; when the chondroitin sulfate polysaccharide is degraded, the final products are a series of unsaturated oligosaccharides, including but not limited to unsaturated disaccharide to unsaturated dodecanose; when the heparin is degraded, the final product is mainly unsaturated disaccharide; when dermatan sulfate is degraded, a final product comprises unsaturated tetrasaccharide to unsaturated octasaccharide. The chondroitin sulfate lyase CHa3 disclosed by the invention can be applied to preparation of CS / DS oligosaccharides and research on CS / DS structures and functions, and has a wide application prospect in the fields of medicines, foods and the like.

Description

Technical Field

[0001] The invention relates to a PL35 family chondroitin sulfate lyase CHa3 and a coding gene and application thereof, belonging to the technical field of enzyme engineering. Background Art

[0002] Glycosaminoglycans (GAGs) are a class of polyanionic straight-chain acidic polysaccharides that are often distributed in the extracellular matrix (ECM) and cell surfaces and participate in a variety of physiological and pathological processes, such as cell growth, cell adhesion, cell proliferation, tissue morphogenesis, cell signal transduction, viral infection, etc. Glycosaminoglycans are composed of repeated alternations of the basic disaccharide units hexuronic acid (D-glucuronic acid [GlcA] or L-iduronic acid [IdoA]) and hexosamine (D-galactosamine [GalNAc] or D-glucosamine [GlcNAc]). These disaccharide repeating units can also be N- or O-sulfated to form sulfated glycosaminoglycans with diverse structures and complex functions. Based on the differences in disaccharide composition, GAGs can be divided into four categories: hyaluronic acid (HA), chondroitin sulfate / dermatan sulfate (CS / DS), heparin / heparan sulfate (HP / HS) and keratin sulfate (KS) (the KS disaccharide unit is composed of D-galactose and D-glucosamine).

[0003] The disaccharide units of CS / DS have a complex sulfation pattern, which increases the complexity of its structure and function. During or after the synthesis of the polysaccharide chain of CS / DS, sulfate groups are often introduced at the C-2 position of GlcA / IdoA and the C-4 and C-6 positions of GalNAc due to the action of various enzymes, such as N-deacetylase-N-sulfotransferase and sulfotransferase; GlcA is converted to IdoA under the action of C5 epimerase. Based on these modifications, CS synthesized in nature is usually composed of six disaccharides with different sulfation patterns: the non-sulfated O / iO unit (-GlcA / IdoA-GalNAc-), the A / iA unit sulfated at the 4-hydroxyl group of GalNAc (-GlcA / IdoA-GalNAc4S-), the C / iC unit sulfated at the 6-hydroxyl group of GalNAc (-GlcA / IdoA-GalNAc6S-), the D / iD unit disulfated at the 2-hydroxyl group of GlcA / IdoA and the 4-hydroxyl group of GalNAc (-GlcA / IdoA2S-GalNAc6S-), the E / iE unit disulfated at the 4- and 6-hydroxyl groups of GalNAc (-GlcA / IdoA-GalNAc4S,6S-), and the T / iT unit trisulfated at the 2-hydroxyl group of GlcA / IdoA and the 4- and 6-hydroxyl groups of GalNAc. unit(-GlcA / IdoA2S-GalNAc4S,6S-). In addition, iB unit(-IdoA2S-GalNAc4S-) is often found in DS. As a variety of disaccharide units are shown in CS / DS, the polysaccharide structure becomes more complex and exhibits many important biological functions.

[0004] The high complexity of GAGs structure and function has brought challenges to the study of GAGs structure-activity relationship. Tool enzymes with specific activities are often used to solve the problems related to GAGs structure-activity relationship research. According to the degradation mechanism of the enzyme, GAGs degrading enzymes can be divided into hydrolases and lyases, which are widely present in animals and microorganisms. GAGs hydrolases cut glycosidic bonds and introduce a molecule of water, while GAGs lyases cut glycosidic bonds and introduce unsaturated double bonds at the C4 and C5 positions of uronic acid. Common GAGs lyases include HA lyase (degrading HA), chondroitin sulfate lyase (degrading HA and CS / DS), and heparinase (degrading HP / HS), each of which shows different substrate preferences. The GAGs lyase family includes the HA lyase and chondroitin sulfate lyase (CSase) families, such as PL6, PL8, PL16, PL23, PL29, PL30, PL33 and PL35, and the heparinase family, such as PL12, PL13, PL21 and PL15. In general, lyases (including HA lyases, CSases, and heparinases) show relatively conservative substrate selectivity. For example, CSases can degrade HA and CS / DS, but not HP / HS, while heparinase can degrade HP / HS, but not HA or CS / DS. The PL35 family is a recently established family of polysaccharide lyases that belongs to the chondroitin sulfate lyase family, but detailed studies and related applications of this family are rare. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a PL35 family chondroitin sulfate lyase CHa3 and its encoding gene and application, wherein the enzyme has highly efficient enzymatic activity for degrading chondroitin sulfate.

[0006] The technical solution of the present invention is as follows:

[0007] A chondroitin sulfate lyase CHa3, whose amino acid sequence is shown in SEQ ID NO.2.

[0008] The chondroitin sulfate lyase CHa3 of the present invention is an endoenzyme. When the chondroitin sulfate lyase CHa3 degrades chondroitin sulfate polysaccharide, the final product is a series of unsaturated oligosaccharides, including but not limited to unsaturated disaccharides to unsaturated dodecasaccharides; when the chondroitin sulfate lyase CHa3 degrades heparin, the final product is an unsaturated disaccharide; when the chondroitin sulfate lyase CHa3 degrades dermatan sulfate, the final product is an unsaturated tetrasaccharide to an unsaturated octasaccharide; the recombinant chondroitin sulfate lyase CHa3 cannot degrade hyaluronic acid and heparan sulfate.

[0009] A coding gene that can encode the above-mentioned chondroitin sulfate lyase Cha3.

[0010] Preferably according to the present invention, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.1.

[0011] A recombinant expression vector is provided, in which the coding gene of the chondroitin sulfate lyase CHa3 is inserted.

[0012] Preferably according to the present invention, the expression vector is an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a bacteriophage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector.

[0013] A recombinant cell, in which the coding gene of the chondroitin sulfate lyase CHa3 or the recombinant expression vector is inserted into the host cell.

[0014] Preferably according to the present invention, the host cell is an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell or a mammalian cell.

[0015] Further preferably, the Escherichia coli host cell is Escherichia coli BL 21, Escherichia coli JM109 or Escherichia coli DH 5α; the yeast host cell is Saccharomyces cerevisiae, Pichia pastoris or Kluyveromyces lactis; the Bacillus subtilis host cell is Bacillus subtilis R25 or Bacillus subtilis9920; the lactic acid bacteria host cell is Lactic acid bacteria COCC101; the actinomycete host cell is Streptomyces spp.; the filamentous fungal host cell is Trichoderma viride, Trichoderma reesei, Aspergillus niger or Aspergillus nidulans; the insect cell is Bombyxmori or Antharaea eucalypti; the mammalian cell is Chinese hamster ovary cell CHO, baby hamster kidney cell BHK or Chinese hamster lung cell CHL.

[0016] The use of the above recombinant cells in the recombinant expression of the above chondroitin sulfate lyase CHa3.

[0017] The application of the above chondroitin sulfate lyase CHa3 in the preparation of CS / DS oligosaccharides.

[0018] The application of the above-mentioned chondroitin sulfate lyase CHa3 in studying the structure and function of CS / DS.

[0019] Beneficial effects:

[0020] 1. The chondroitin sulfate lyase CHa3 in the present invention is isolated from the sea mud metagenomic library, is easy to heterologously express and purify, has stable physicochemical properties, high degradation activity, and has the potential for industrial application.

[0021] 2. The chondroitin sulfate lyase CHa3 in the present invention is a new type of chondroitin sulfate lyase, which has a high enzyme activity for chondroitin sulfate. The enzyme activities for chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, and chondroitin sulfate E are 1.11 U / mg, 4.78 U / mg, 2.63 U / mg, and 1.91 U / mg, respectively.

[0022] 3. The chondroitin sulfate lyase CHa3 in the present invention degrades chondroitin sulfate in an endoenzyme mode; when degrading chondroitin sulfate polysaccharides, the final product is a series of unsaturated oligosaccharides, including but not limited to unsaturated disaccharides to unsaturated dodecasaccharides; when degrading heparin, the final product is mainly unsaturated disaccharides; when degrading dermatan sulfate, the final product includes unsaturated tetrasaccharides to unsaturated octasaccharides. The chondroitin sulfate lyase CHa3 in the present invention can be applied to the preparation of CS / DS oligosaccharides and the study of CS / DS structure and function, and has broad application prospects in the fields of medicine and food.

[0023] 4. When the chondroitin sulfate lyase CHa3 of the present invention degrades chondroitin sulfate A, a series of unsaturated oligosaccharides are produced, including unsaturated disaccharides, unsaturated tetrasaccharides, unsaturated hexasaccharides, unsaturated octasaccharides, unsaturated decasaccharides, and unsaturated dodecasaccharides. The disaccharide composition analysis results of the above unsaturated oligosaccharides show that CHa3 belongs to a typical chondroitin sulfate lyase C, and the final product oligosaccharides of CSA degradation are composed of O, A and C units, and the number of A and C units decreases with the increase of oligosaccharide size. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a functional module analysis diagram of chondroitin sulfate lyase CHa3.

[0025] Figure 2 This is the evolutionary tree analysis diagram of chondroitin sulfate lyase CHa3.

[0026] Figure 3 This is the protein three-dimensional structure model of chondroitin sulfate lyase CHa3.

[0027] Figure 4The polyacrylamide gel electrophoresis pattern of the expression and purification samples of chondroitin sulfate lyase CHa3; wherein: lane 1, protein molecular weight standard, sample volume 5 μL, the sizes of the bands from top to bottom are 180kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa; lane 2, bacterial solution after cell wall breaking of control strain, sample volume 8 μL; lane 3, bacterial solution after cell wall breaking of recombinant strain, sample volume 5 μL; lane 4, supernatant after cell wall breaking of recombinant strain, sample volume 4 μL; lane 5, CHa3 enzyme solution purified by nickel column, sample volume 5 μL.

[0028] Figure 5 This is the effect curve of temperature on the activity of chondroitin sulfate lyase CHa3.

[0029] Figure 6 This is the effect curve of pH on the activity of chondroitin sulfate lyase CHa3.

[0030] Figure 7 This is the curve showing the effect of temperature on the stability of chondroitin sulfate lyase CHa3.

[0031] Figure 8 This is a bar graph showing the effects of metal ions and other compounds on the activity of chondroitin sulfate lyase CHa3.

[0032] Fig. 9 This is the effect curve of NaCl concentration on the activity of chondroitin sulfate lyase CHa3.

[0033] Fig.10 HPLC chromatograms of degradation products of different substrates degraded by chondroitin sulfate lyase CHa3; in the figure: Figure A is an HPLC chromatogram with hyaluronic acid (HA) as substrate, Figure B is an HPLC chromatogram with chondroitin sulfate A (CSA) as substrate, Figure C is an HPLC chromatogram with chondroitin sulfate C (CSC) as substrate, Figure D is an HPLC chromatogram with chondroitin sulfate D (CSD) as substrate, Figure E is an HPLC chromatogram with chondroitin sulfate E (CSE) as substrate, Figure F is an HPLC chromatogram with dermatan sulfate (DS) as substrate, Figure G is an HPLC chromatogram with heparin (HP) as substrate, and Figure H is an HPLC chromatogram with heparan sulfate (HS) as substrate; wherein, Di-1S, monosulfated GAGs unsaturated disaccharide; Tetra, GAGs unsaturated tetrasaccharide; Hexa, GAGs unsaturated hexasaccharide; Octa, GAGs unsaturated octasaccharide; Deca, GAGs unsaturated decasaccharide; Dodeca, GAGs unsaturated dodecasaccharide.

[0034] Fig.11The HPLC chromatograms show the degradation products of CSD at different times after degradation by chondroitin sulfate lyase CHa3; in the figure: Di-1S, monosulfated CSD unsaturated disaccharide; Tetra, CSD unsaturated tetrasaccharide; Hexa, CSD unsaturated hexasaccharide; Octa, CSD unsaturated octasaccharide; Deca, CSD unsaturated decasaccharide; Dodeca, CSD unsaturated dodecasaccharide.

[0035] Fig.12 HPLC chromatograms for disaccharide composition analysis of the final product of CSA degradation by chondroitin sulfate lyase CHa3: Figure A is an HPLC chromatogram of unsaturated disaccharides of the final product; Figure B is an HPLC chromatogram of the disaccharide composition of unsaturated tetrasaccharides of the final product; Figure C is an HPLC chromatogram of the disaccharide composition of unsaturated hexasaccharides of the final product; Figure D is an HPLC chromatogram of the disaccharide composition of unsaturated octasaccharides of the final product; Figure E is an HPLC chromatogram of the disaccharide composition of unsaturated decasaccharides of the final product; Figure F is an HPLC chromatogram of the disaccharide composition of unsaturated dodecasaccharides of the final product. DETAILED DESCRIPTION

[0036] The following examples are provided to fully disclose some common techniques for implementing the present invention, rather than to limit the scope of application of the present invention. The inventors have made every effort to ensure the accuracy of each parameter in the examples (e.g., amount, temperature, etc.), but some experimental errors and deviations should also be considered. Unless otherwise specified, molecular weight refers to weight-average molecular weight, and temperature refers to degrees Celsius in the present invention. The reagents and materials involved in the examples are all common commercially available products unless otherwise specified.

[0037] Sources of biological materials:

[0038] The sea mud samples were collected from the Hutouya intertidal zone in Yantai, Shandong (N37°18'31", E119°09'27"), and the samples were collected on July 8, 2021.

[0039] Example 1: Establishment of a metagenomic library

[0040] The intertidal mud samples were added to the sole carbon source medium and cultured at 28°C and 200 rpm for 3 days. One milliliter of the bacterial solution was transferred to fresh sole carbon source medium and then cultured at 28°C and 200 rpm for 3 days. The cultured bacteria were collected by centrifugation at 8000×g for 20 minutes, and the metagenomic DNA was sequenced and annotated at BGI Technology Service Co., Ltd. (Guangdong, China) to construct the metagenomic library.

[0041] The above-mentioned sole carbon source medium has the following components per liter:

[0042] K2HPO4 1g, MgSO4·7H2O 0.5g, NaCl 30g, CaCl2·2H2O 0.1g, FeSO4 0.001g, NH4Cl1g, chondroitin sulfate C 5g, water 1000mL, pH 7.0.

[0043] Example 2: Extraction of bacterial DNA

[0044] Take 40 mL of the culture solution of Example 1, centrifuge at 12,000 rpm for 25 min, collect the bacterial precipitate, wash with 20 mL of lysozyme buffer (10 mM Tris-HCl, pH 8.0), centrifuge at 12,000 rpm for 25 min, and collect the bacterial precipitate;

[0045] 12.0 mL of lysozyme buffer (10 mM Tris-HCl, pH 8.0) was added to the above bacterial precipitate to obtain about 14.0 mL of bacterial solution, and 560 μL of lysozyme solution with a concentration of 20 mg / mL was added, with a final concentration of about 800 μg / mL; after an ice bath for 1.0 h, it was warmed at 37°C for 2 h until the solution became viscous; 0.82 mL of 10 wt% SDS (sodium dodecyl sulfate) solution and 60 μL of 100 mg / mL proteinase K solution were added, and the mixture was bathed at 52°C for 1.0 h; 15 mL of Tris-equilibrated phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) was added, and the mixture was gently inverted to mix until fully emulsified; centrifuged at 10,000 × g and 4°C for 10 min, the supernatant was transferred, and 2.0 mL of NaAc-HAc buffer (pH 5.2, 3.0M), and 17.0mL of anhydrous ethanol, mix well; pick out the filamentous DNA with a 1.0mL pipette tip, transfer to a 1.5mL EP centrifuge tube, wash twice with 70wt% ethanol (stored at -20℃), discard the supernatant after microcentrifugation; centrifuge at 10,000×g, 4℃ for 3min, and completely discard the supernatant; blow dry the sample under an alcohol lamp on a sterile workbench; resuspend and dissolve the DNA sample with sterile deionized water, and store at 4℃ overnight to obtain high molecular weight genomic DNA.

[0046] Example 3: Sequence analysis of chondroitin sulfate lyase CHa3

[0047] The metagenomic library prepared in Example 1 was sequenced using software from NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ). The NCBI analysis software used was Open Reading Frame Finder (ORF Finder, http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0048] The results of NCBI analysis showed that the chondroitin sulfate lyase gene cha3 in the metagenomic library had a gene coding region length of 1884bp, and its nucleotide sequence was shown in SEQ ID NO.1. The chondroitin sulfate lyase CHa3 encoded by the chondroitin sulfate lyase gene cha3 was composed of 627 amino acids, and its amino acid sequence was shown in SEQ ID NO.2. The theoretical molecular weight of the protein was about 69.25kDa. The results of online analysis using BLAST software showed that the chondroitin sulfate lyase CHa3 had 28.84% homology with the chondroitin sulfate lyase chondroitinase (NCBI sequence number: WP_116885688.1) composed of 792 amino acids encoded by the heparinase II / III family protein gene in the whole genome sequence of Victivallis vadensis, which had been studied for its activity.

[0049] The structural information of chondroitin sulfate lyase CHa3 was analyzed using Simple Modular Architecture Research Tool (SMART, http: / / smart.embl-heidelberg.de / ). The results showed that the 1st to 35th amino acids at the N-terminus were signal peptide sequences, and the 416th to 555th amino acid sequences belonged to the Hepar-II-III family. Figure 1 As shown. By analyzing CHa3 with other types of chondroitin sulfate lyases that have been identified, CHa3 is in the same evolutionary branch as the PL35 family, as shown in Figure 2The homology modeling of the protein three-dimensional structure of chondroitin sulfate lyase CHa3 was performed using the SWISS-MODEL homology modeling server (http: / / swissmodel.expasy.org). The final three-dimensional structure model of the protein of chondroitin sulfate lyase CHa3 is shown in Figure 3 shown.

[0050] Example 4: Recombinant expression of cha3 gene in Escherichia coli

[0051] The high molecular weight genomic DNA prepared in Example 2 was used as a template for PCR amplification, and the primers were as follows:

[0052] Forward primer cha3-F: 5′-ATCTGTATACATATGGCGATGGATACGGTGAAATTGGAG-3′ (SEQ ID NO. 3);

[0053] Reverse primer cha3-R: 5′-GTGGTGGTGCTCGAGCAATGCTTTTAATCTAACCTTGATTTTC-3′ (SEQ ID NO. 4);

[0054] PrimeSTAR HSDNA polymerase was purchased from Takara Biotech, and the PCR reaction system was operated according to the product instructions provided by the company.

[0055] The reaction conditions of PCR amplification were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, 35 cycles; extension at 72°C for 10 min, and stabilization at 4°C for 15 min.

[0056] After the cha3 gene fragment was amplified by PCR, the PCR amplification product, pET-30a expression vector (the vector was linearized by double restriction enzymes NdeI and XhoI) and recombinase Exnase II were prepared into a recombination reaction system. The recombination reaction was completed by reacting at 37°C for 30 minutes to achieve in vitro cyclization of the two linear DNAs. The recombinant product was directly transformed into the Escherichia coli DH5α (purchased from Nanjing Novozymes) strain, and then spread on an LB solid culture medium plate containing 50 μg / mL kanamycin, and cultured at 37°C for 12-14 hours. A single clone was picked and transferred to an LB liquid culture medium containing 50 μg / mL kanamycin. After culturing at 37°C and 200 rpm on a shaking table for 12-14 hours, the forward primer cha3-F and the reverse primer cha3-R were used to verify the bacterial liquid PCR. As a result, an amplification product of the correct size was obtained, which preliminarily proved that the constructed recombinant plasmid was correct. 20 μL of the recombinant plasmid was taken out and sent to Sangon Biotech for sequencing. The sequencing results showed that the cha3 gene fragment (SEQ ID NO.1) was successfully inserted into the restriction sites of the pET-30a expression vector, the insertion direction was correct, and no base mutation, deletion or addition occurred, so it was further proved that the constructed recombinant plasmid was correct, and the recombinant plasmid was named pET30a-cha3. The recombinant enzyme Exnase II was purchased from Bao Bio, and the pET-30a expression vector was purchased from Novagen, USA. The reaction system, reaction temperature and reaction time of the enzyme and substrate were all in accordance with the instruction manual.

[0057] The recombinant plasmid pET30a-cha3 was transformed into Escherichia coli BL21 (DE3) (purchased from Nanjing Novozyme Co., Ltd.), and then the recombinant chondroitin sulfate lyase CHa3 was induced to express according to the operation steps provided by the company. The target protein was purified by NiSepharose 6 Fast Flow (GE) gel to obtain CHa3 enzyme solution, and the purified target protein was detected by polyacrylamide gel electrophoresis. The detection results are shown in FIG. Figure 4 As shown, the purified chondroitin sulfate lyase CHa3 showed a single band on the electrophoresis gel, and the position was consistent with the predicted molecular weight, and the purity reached 95%.

[0058] Example 5: Analysis of Enzymatic Properties of Recombinant Chondroitin Sulfate Lyase CHa3

[0059] 1. Effect of temperature on enzyme activity

[0060] First, 10 mg / mL chondroitin sulfate C solution, 150 mM pH 8.0 NaH2PO4-Na2HPO4 buffer, CHa3 enzyme solution and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio), and reacted at 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C and 70°C for 30 min. After the reaction, the enzyme activity was determined by UV spectrophotometry, and the best enzyme activity was defined as 100% relative activity. The test results are shown in Figure 5 As shown, the relative enzyme activity of CHa3 towards chondroitin sulfate C is above 90% at 20-30°C, and the enzyme activity of CHa3 decreases rapidly when the reaction temperature is above 40°C; among them, CHa3 reaches the maximum activity towards chondroitin sulfate C at 20°C, indicating that the optimal reaction temperature of recombinant chondroitin sulfate lyase CHa3 towards chondroitin sulfate C is 20°C.

[0061] The method of determining enzyme activity by ultraviolet spectrophotometry refers to the prior art (Yamagata, T., et al., Purification and properties of bacterial chondroitinases and chondrosulfatases. The Journal of biological chemistry, 1968. 243 (7): p. 1523-35), which uses inactivated enzyme as negative control and uses a spectrophotometer to measure the light absorption of the reaction product at 232 nm to determine the product production and thus the size of the enzyme activity.

[0062] 2. Effect of pH on enzyme activity

[0063] 10mg / mL chondroitin sulfate C solution, reaction buffer, CHa3 enzyme solution and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio). The reaction buffer included 150mM NaAc-Hac buffer (pH5.0-6.0), 150mM NaH2PO4-Na2HPO4 buffer (pH6.0-8.0), and 150mM Tris-HCl buffer (pH7.0-10.0). The reaction was carried out at the optimum temperature for 30min. After the reaction was completed, the enzyme activity was determined by ultraviolet spectrophotometry. The best enzyme activity was defined as 100% relative activity. The test results are as follows: Figure 6 As shown, the recombinant chondroitin sulfate lyase CHa3 has the highest enzyme activity towards chondroitin sulfate C in NaH2PO4-Na2HPO4 buffer (pH 8.0).

[0064] 3. Effect of temperature on enzyme stability

[0065] The residual enzyme activity of CHa3 enzyme solution and 10 mg / mL chondroitin sulfate C solution after heat treatment at different temperatures (0°C-70°C) for 1, 2, 4, 8, 12, and 24 hours was measured at the optimal temperature and optimal pH. The enzyme activity of CHa3 enzyme solution before heat treatment was defined as 100% relative activity. The test results are shown in Figure 7 As shown, after being treated at 0-20°C for 24 hours, the recombinant chondroitin sulfate lyase CHa3 still maintains more than 80% relative enzyme activity for chondroitin sulfate C; after being treated at an environment above 30°C for 24 hours, the relative enzyme activity of the recombinant chondroitin sulfate lyase CHa3 for chondroitin sulfate C drops sharply, indicating that temperature has a great influence on the stability of CHa3 enzyme and it is not resistant to high temperature.

[0066] 4. Effects of metal ions on enzyme activity

[0067] 10 mg / mL chondroitin sulfate C solution, 150 mM NaH2PO4-Na2HPO4 buffer solution at pH 8.0, CHa3 enzyme solution and deionized water were mixed in a ratio of 10:10:3:4 (volume ratio). Then different metal ions or compounds were added to the reaction system with a final concentration of 5 mM. The reaction was carried out at the optimal temperature for 30 min. After the reaction was completed, the residual enzyme activity was detected by ultraviolet spectrophotometry. The enzyme activity without adding metal ions was defined as 100% relative activity. The test results are shown in FIG. Figure 8 As shown in the figure, metal ions have a strong promoting effect on the enzyme activity of CHa3, while Fe 3+ 、Zn 2+ , β-mercaptoethanol, DTT, Ni 2+ The enzyme activity of CHa3 was slightly inhibited by Ag. + 、Cd 2+ , Cu 2+ , Hg 3+ Cr 3+ , Fe 2 + , EDTA and SDS had a strong inhibitory effect on the enzyme activity of CHa3.

[0068] 5. Effect of NaCl concentration on enzyme activity

[0069] 10mg / mL chondroitin sulfate C solution, 150mM pH8.0 NaH2PO4-Na2HPO4 buffer, CHa3 enzyme solution and deionized water were mixed in a ratio of 10:10:3:4 (volume ratio), and then different final concentrations of NaCl were added to the reaction system. The final concentrations of NaCl added were 0mM, 1mM, 2mM, 5mM, 10mM, 20mM, 50mM, 100mM, 250mM, 500mM and 1000mM, respectively. The reaction was carried out at the optimal temperature for 30min. After the reaction, the residual enzyme activity was detected by ultraviolet spectrophotometry. The enzyme activity with a final NaCl concentration of 0mM was defined as 100% relative activity. The test results are as follows: Fig. 9 As shown in the figure, with the increase of NaCl concentration, the enzyme activity of CHa3 gradually decreased, indicating that NaCl has an inhibitory effect on the enzyme activity of CHa3.

[0070] Example 6: Determination of the enzyme activity of recombinant chondroitin sulfate lyase CHa3

[0071] 10mg / mL chondroitin sulfate C solution, 150mM pH8.0 NaH2PO4-Na2HPO4 buffer, CHa3 enzyme solution and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio), and reacted at the optimum temperature for 1-10min. An equal amount of inactivated enzyme was added to the negative control. After the reaction, the enzyme activity was determined by the aforementioned UV spectrophotometry.

[0072] The enzyme activities of chondroitin sulfate A, chondroitin sulfate D, and chondroitin sulfate E were determined according to the above method.

[0073] Definition of enzyme activity unit (U): the amount of enzyme required to generate 1 μmol of product containing unsaturated double bonds per minute.

[0074] At the same time, the protein content of CHa3 enzyme solution was determined using a protein quantification kit purchased from Kangwei Century Company. The results showed that the enzyme activities of recombinant chondroitin sulfate lyase CHa3 for chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, and chondroitin sulfate E were 1.11U / mg, 4.78U / mg, 2.63U / mg, and 1.91U / mg, respectively.

[0075] Example 7: High Performance Liquid Chromatography (HPLC) Analysis of Degradation Products of Different Substrates by Recombinant Chondroitin Sulfate Lyase CHa3

[0076] A 10 mg / mL substrate (chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, chondroitin sulfate E, dermatan sulfate, heparin, heparan sulfate or hyaluronic acid) solution, 150 mM NaH2PO4-Na2HPO4 buffer (pH 8.0), CHa3 enzyme solution and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio), reacted overnight at the optimal temperature, and the degradation products were analyzed by HPLC. The HPLC analysis conditions were: gel column: Superdex peptide 10 / 300GL (GE); mobile phase: 0.2 M ammonium bicarbonate; flow rate: 0.4 mL / min; detection conditions: UV 232 nm.

[0077] Test results such as Fig.10 As shown, when the recombinant chondroitin sulfate lyase CHa3 degrades chondroitin sulfate polysaccharide, the final product is a series of unsaturated oligosaccharides, including but not limited to unsaturated disaccharides to unsaturated dodecasaccharides; when the recombinant chondroitin sulfate lyase CHa3 degrades heparin, the final product is an unsaturated disaccharide; when the recombinant chondroitin sulfate lyase CHa3 degrades dermatan sulfate, the final product includes unsaturated tetrasaccharides to unsaturated octasaccharides; the recombinant chondroitin sulfate lyase CHa3 cannot degrade hyaluronic acid and heparan sulfate.

[0078] Example 8: Degradation pattern of recombinant chondroitin sulfate lyase CHa3

[0079] 10 mg / mL CSD solution, CHa3 enzyme solution, 150 mM NaH2PO4-Na2HPO4 buffer (pH 8.0) and deionized water were mixed in a ratio of 10:3:10:7 (volume ratio), reacted at the optimal temperature, and the degradation products of different enzymatic hydrolysis times were selected for HPLC analysis. The HPLC analysis conditions were: gel column: Superdex peptide 10 / 300GL (GE); mobile phase: 0.2 M ammonium bicarbonate; flow rate: 0.4 mL / min; detection conditions: UV 232 nm.

[0080] Test results such as Fig.11 As shown, when CHa3 degrades CSD, a large amount of large molecular weight unsaturated oligosaccharides are first generated, and then the large molecular weight oligosaccharides are gradually degraded into smaller molecular weight oligosaccharides, and finally a series of unsaturated oligosaccharides are generated, including but not limited to unsaturated disaccharides to unsaturated dodecasaccharides. This result indicates that the recombinant chondroitin sulfate lyase CHa3 is an endo-enzyme.

[0081] Example 9: Analysis of oligosaccharide composition of the final product of CSA degradation by recombinant chondroitin sulfate lyase CHa3

[0082] 10mg / mL chondroitin sulfate A solution, 150mM NaH2PO4-Na2HPO4 buffer (pH8.0), CHa3 enzyme solution and deionized water were mixed in a ratio of 10:2:3:15 (volume ratio), reacted overnight at the optimal temperature, and the degradation products were analyzed by HPLC. HPLC analysis conditions are: gel column: Superdex peptide 10 / 300GL (GE); mobile phase: 0.2M ammonium bicarbonate; flow rate: 0.4mL / min; detection conditions: UV 232nm. The unsaturated disaccharides, unsaturated tetrasaccharides, unsaturated hexasaccharides, unsaturated octasaccharides, unsaturated decasaccharides and unsaturated dodecasaccharides of the final product were recovered and freeze-dried three times to remove salt.

[0083] 2 μg / mL of each component solution of the final product, 150 mM NaH2PO4-Na2HPO4 buffer (pH 8.0), CSaseABC enzyme solution and deionized water were mixed in a ratio of 10:2:3:15 (volume ratio), reacted overnight under optimal conditions, and the degradation products were 2-AB labeled and then analyzed by HPLC. HPLC analysis conditions are: ion column: CarboPac PA1; mobile phase: 0.05-2M sodium chloride; flow rate: 1 mL / min; detection conditions: fluorescence detector, excitation light 330 nm, emission light 420 nm.

[0084] The test results showed that the disaccharide compositions of the final products of oligosaccharide unsaturated disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, decasaccharide and dodecasaccharide of CSA degradation by recombinant chondroitin sulfate lyase CHa3 were ΔC, ΔO / ΔA / ΔC (molar ratio of 1:3:12), ΔO / ΔA / ΔC (molar ratio of 1:1.62:3.5), ΔO / ΔA / ΔC (molar ratio of 1:1.6:2.1), ΔO / ΔA / ΔC (molar ratio of 1:1.9:1.5) and ΔO / ΔA / ΔC (molar ratio of 1:2:1.3) respectively. Fig.12 ). These results indicate that the only disaccharide product produced by CHa3 when degrading the substrate is ΔC, which is a typical characteristic of chondroitin sulfate lyase C (mainly degrading the C unit segment in CS). In addition, the oligosaccharide products are composed of three disaccharide units: ΔO / ΔA / ΔC, and the proportion of ΔA gradually increases with the increase of oligosaccharide size, which means that CHa3 tends to degrade the ΔC aggregation segment and cannot degrade other regions other than those containing ΔC.

Claims

1. A chondroitin sulfate lyase CHa3, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

2. A coding gene, characterized in that The chondroitin sulfate lyase Cha3 according to claim 1 can be encoded.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown as SEQ ID NO.

1.

4. A recombinant expression vector, characterized in that: The coding gene according to claim 2 is inserted into the expression vector.

5. The recombinant expression vector according to claim 4, characterized in that The expression vector is an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a bacteriophage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector.

6. A recombinant cell, characterized in that The encoding gene according to claim 2 or the recombinant expression vector according to claim 4 is inserted into the host cell.

7. The recombinant cell according to claim 6, characterized in that The host cell is an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell or a mammalian cell; Further preferably, the Escherichia coli host cell is Escherichia coli BL 21, Escherichia coli JM 109 or Escherichia coli DH 5α; the yeast host cell is Saccharomyces cerevisiae, Pichia pastoris or Kluyveromyces lactis; the Bacillus subtilis host cell is Bacillus subtilis R25 or Bacillus subtilis 9920; the lactic acid bacteria host cell is Lactic acidbacteria COCC101; the actinomycete host cell is Streptomyces spp.; the filamentous fungal host cell is Trichoderma viride, Trichoderma reesei, Aspergillus niger or Aspergillus nidulans; the insect cell is Bombyx mori or Antharaea eucalypti; the mammalian cell is Chinese hamster ovary cell CHO, baby hamster kidney cell BHK or Chinese hamster lung cell CHL.

8. Use of the recombinant cell according to claim 6 in recombinantly expressing the chondroitin sulfate lyase CHa3 according to claim 1.

9. Use of the chondroitin sulfate lyase CHa3 according to claim 1 in the preparation of CS / DS oligosaccharides.

10. Use of the chondroitin sulfate lyase CHa3 according to claim 1 in studying the structure and function of CS / DS.

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